Study on the biosynthesis of the modified uridine derivatives at the first position of the anticodon of tRNA

1. 在抗生素培养基的IOL中培养枯草芽孢杆菌3 120次。以88%苯酚为溶剂从枯草芽孢杆菌细胞中提取粗tRNA，并采用多种柱层析法进行纯化。纯化了甘氨酸（2种）、赖氨酸、苏氨酸、缬氨酸接受trna。为了用未修饰的寡核苷酸改变反密码子核苷酸，每个tRNA分子分别用RNase TI、RNase A或核酸酶Sl酶切，用聚丙烯酰胺凝胶电泳纯化3′和5′半分子。RNase H对于获得tRNA的特定半分子是非常有效的。这种酶可以在RNA=DNA的双链区域分裂成RNA链。合成了与反密码子茎和环区核苷酸序列互补的嵌合低聚物。永久性U34和C33之间的磷酸二酯键被特异性水解。通过RNA连接酶将甘氨酸、赖氨酸、苏氨酸trna中的5′-半分子与未修饰的寡核苷酸[32P]pUp、[32P]pUUU或[32P]pUA连接。3′端磷酸被pase消除后，如果有，则用相应的3′半分子进行退火处理。通过RNA连接酶将两个半段再次连接，得到一个包含未修饰的反密码子核苷酸的完整tRNA分子。重建的tRNA分子与枯草芽孢杆菌S-100或S-30组分孵育。孵育后，用醇沉淀tRNA，用核酸酶Pl水解。用薄层色谱法检查反密码子第1位尿苷酸的修饰，该反密码子在5'磷酸处含有[32P]。重组的tRNA分子在反密码子的第一个核苷酸处未被粗酶部分修饰。由于底物或共底物的浓度低，同位素标记的tRNA可能难以修饰为底物。这两年来，我们在这个项目上没有取得多大进展。但是，我们希望继续这个研究项目，以获得预期的结果。少

1. Bacillus subtilis was cultivated in IOL of a medium of Antibiotic Medium 3 120 times. Crude tRNA was extracted from B. subtilis cells with 88% phenol and purified by a combination of several column chromatography. Glycine(2 kinds),lysine, threonine, valine accepting tRNAs were purified.2. In order to change the anticodon nucleotides with unmodified oligonucleotide, each tRNA molecule was digested with RNase TI, RNase A or nuclease Sl. 3'- and 5' half molecules were purified by polyacrylamide gel electrophoresis.3. RNase H was very effective to obtain the specific half molecule of tRNA. The enzyme can split in RNA strand in the double strand region of RNA=DNA. The chimeric oligomers which is complimentary to the nucleotide sequence of stem and loop region of anticodon were synthesized. The phosphodiester bond between permanent U34 and C33 was specifically hydrolyzed.4. 5'-Half molecules from glycine, lysine, threonine tRNAs were ligated with the unmodified oligonucleotide, [32P]pUp, … More [32P]pUUU or [32P]pUA by RNA ligase. After 3'-terminal phosphate was eliminated by PMase, if any, they were annealed with corresponding 3'-half molecule. Two halves were ligated again by RNA ligase to give a whole tRNA molecule which contains unmodified anticodon nucleotide.5. Reconstructed tRNA molecules were incubated with B. subtilis S-100 or S-30 fraction. After incubation, tRNA was precipitated by alcohol and hydrolyzed with nuclease Pl. The modification of uridylic acid in the first position of the anticodon, which contains [32P] at the 5' phosphate was checked by thin layer chromatography. None of the reconstructed tRNA molecule was modified at the first nucleotide in the anticodon by crude enzyme fraction. It may be difficult to modify isotope-labeled tRNA as a substrate because of the low concentration of a substrate or cosubstrate.6. We have not obtained a much progress in this project in these two years. But, we hope to continue this research project to get an anticipated results. Less